La fibrose kystique (FK), causée par des mutations dans le gène codant pour le canal chlorure CFTR, est caractérisée par des infections bactériennes chroniques des voies aériennes (VA), impliquant en particulier Pseudomonas aeruginosa (PA) et Staphylococcus aureus (SA). Les facteurs de virulence (VirF) sécrétés par celles-ci sont responsables de la destruction progressive des VA et altèrent la capacité de réparation du tissu épithélial. Il existe cependant des molécules spécifiques permettant de corriger le défaut de CFTR et de moduler l’activité de canaux potassiques, deux actions qui pourraient favoriser la réparation épithéliale de par leur action. Leur efficacité pourrait toutefois être altérée par les VirF de P. aeruginosa ou S. aureus. Mon objectif a été d’identifier le potentiel de réparation épithélial du Trikafta et du ML277, modulateurs respectivement spécifiques des canaux CFTR et KvLQT1 (canal potassique sensible au voltage, du syndrome du QT long), impliqués dans les processus de réparation. Des cultures primaires de cellules bronchiques des VA de patients FK ont été traitées avec la triple combinaison de modulateurs de CFTR Trikafta, l’activateur de KvLQT1 ML277, et la combinaison de ces deux derniers, en plus d’être exposées aux VirF de cultures de P. aeruginosa ou S. aureus. L’efficacité de ces traitements sur les processus de réparation fut évaluée, suite à des lésions, selon la vitesse de réparation des plaies, la prolifération cellulaire et les dynamiques de migration guidée cellulaire. Les VirF de P. aeruginosa et S. aureus altèrent la vitesse de fermeture lésionnelle, la prolifération cellulaire et les dynamiques de migration cellulaire. Les traitements Trikafta et ML277 permettent cependant d’améliorer ces processus de la réparation épithéliale, et ce, en absence comme en présence des VirF bactériens nocifs à la réparation. La combinaison du Trikafta et du ML277 n’amène cependant pas l’effet additif espéré sur la réparation épithéliale. Ces résultats témoignent finalement de l’effet bénéfique du Trikafta et du ML277 sur la réparation épithéliale malgré la condition infectieuse, favorisant l’intégrité fonctionnelle de l’épithélium des VA FK. / Cystic fibrosis (CF), caused by mutations in the gene coding for the chloride channel CFTR, is characterized by chronic bacterial infections in the airways, particularly by Pseudomonas aeruginosa (PA) and Staphylococcus aureus (SA). The virulence factors (VirF) secreted by these bacteria are responsible for the progressive destruction of the airways and impair the repair process of the epithelia. Nevertheless, there are specific molecules that correct the CFTR defect and modulate potassium channel activity, both of which could be beneficial at promoting epithelial repair. However, their efficacy could be altered by P. aeruginosa or S. aureus VirF. My objective was to identify the repair potential of Trikafta and ML277, respectively specific modulators of CFTR and KvLQT1 channels, involved in repair processes. Primary cultures of airways epithelial cells from CF patients were treated with the triple combination of CFTR modulators Trikafta, the KvLQT1 activator ML277, and the combination of the latter two, in addition to being exposed to VirF from P. aeruginosa or S. aureus cultures. The efficacy of these treatments on repair processes was evaluated, following wound injury, by the rate of wound repair, cell proliferation and guided cell migration dynamics. P. aeruginosa and S. aureus VirF alter wound repair rates, cell proliferation and cell migration dynamics. Nevertheless, Trikafta and ML277 treatments improve these epithelial repair processes, both in the absence and presence of repair-damaging P. aeruginosa or S. aureus VirF. However, the combination of Trikafta and ML277 did not have the hoped-for additive effect on epithelial repair. Overall, these results show the beneficial effect of Trikafta and ML277 epithelial repair despite the infectious condition, promoting the functional integrity of the CF airways epithelia.

<p>Systemic fungal infections brought about by <em>Cryptococcus</em> species are associated with some of the highest mortality rates of any infectious disease. Alarmingly these pathogens have overtaken tuberculosis as the second greatest killer among Sub-Saharan AIDS patients and are an emerging disease among immunocompetent populations on the Pacific Coast of North America. This clinical threat has been exacerbated by our inability to discover novel compounds that specifically target fungal cellular architecture at the genus level. To confront this challenge, we have made a concerted effort to biologically prospect the vast chemical potential of Actinomycete bacteria isolated from diverse and underexplored niches around the world. A novel phenotypic screen was developed whereby bacterial small molecule producers were co-cultured on agar plates in an intimate setting with evolutionary distant fungal pathogens <em>Candida albicans</em> and <em>Cryptococcus neoformans</em>. Diffusible small molecules released by the organisms created a signaling environment that stimulated profound phenotypic changes both in the Actinomycetes and the pathogens. We were able to discern a unique relationship whereby the growth of <em>C. neoformans</em> was specifically inhibited by Nigerian soil Actinomycete isolate curated as WAC 2288. Further bioactivity guided purification and chemical analysis lead to the identification of ibomycin, a previously undescribed 34 membered macrolactone decorated with seven sugar moieties. A draft genome of WAC 2288 revealed a 140kb gene cluster containing 12 type I PKS modules and downstream capacity to generate rare sugars are responsible for ibomycin biosynthesis. Purification of ibomycin analogs has revealed that the terminal vancosamine on the molecule is dispensable for bioactivity, establishing a chemical antecedent for target identification through affinity chromatography. Throughout these studies the unprecedented anticryptococcal activity of ibomycin is consistently recapitulated. Future work on the molecule may validate ibomycin as an effective antifungal therapy.</p> / Master of Science (MSc)

Mounting concerns about drug-resistant pathogenic bacteria have rekindled the interest in bacteriophages (bacterial viruses). As bacteria’s natural predators, bacteriophages offer a critical advantage over antibiotics, namely that they can be highly specific. This means that phage therapeutics can be designed to destroy only the infectious agent(s), without causing any harm to our microbiota. However, the potential secondary effects on the balance of microbiota through bacteriophage-induced genome evolution remains as one of the critical apprehensions regarding phage therapy. There exists a significant gap in knowledge regarding the direct and indirect effect of phage therapeutics on the microbiota. The aim of this thesis was to: (1) establish an in vivo model for investigation of the evolutionary dynamics and co-evolution of therapeutic phage and its corresponding host bacterium in the gut; (2) determine if phage therapy can affect the composition of the gut microbiota, (3) observe the differences of phage-resistant bacteria mutants evolved in vivo in comparison to those evolved in vitro. We used germ-free mice colonized with a consortium of eight known bacteria, known as the altered Schaedler flora (ASF). The colonizing strain of choice (mock infection) was a non-pathogenic strain E. coli K-12 (JM83) known to co-colonize the ASF model, which was challenged in vivo with T7 phage (strictly lytic). We compared the composition of the gut microbiota with that of mice not subject to phage therapy. Furthermore, the resistant mutants evolved in vivo and in vitro were characterized in terms of growth fitness and motility. / Thesis / Master of Applied Science (MASc) / Bacteriophages are viruses that infect bacteria. After their discovery in 1917, bacteriophages were a primary cure against infectious disease for 25 years, before being completely overshadowed by antibiotics. With the rise of antibiotic resistance, bacteriophages are being explored again for their antibacterial activity. One of the critical apprehensions regarding bacteriophage therapy is the possible perturbations to our microbiota. We set out to explore this concern using a simplified microbiome model, namely germ-free mice inoculated with only 8 bacteria plus a mock infection challenged with bacteriophage. We monitored this model for 9 weeks and isolated a collection of phage-resistant bacterial mutants from the mouse gut that developed post phage challenge, maintaining the community of mock infection inside the gut. A single dose of lytic phage challenge effectively decreased the mock infection without causing any extreme long-term perturbations to the gut microbiota.

INTRODUÇÃO: Apesar do contínuo progresso no tratamento e suporte clínico a sepse continua sendo uma das principais causas de morbidade e mortalidade nas unidades de terapia intensiva, com desfechos semelhantes ao longo dos últimos 50 anos. A suscetibilidade à infecção grave no recém-nascido é parcialmente devida à imaturidade do sistema imune inato associado à mínima em exposição antigênica in utero e à ação ineficaz das células T efetoras e das célula B. Embora a ativação do sistema imune inato por padrões de reconhecimento (PRR) como os dos receptores Toll-like (TLR) tenham sua importância amplamente reconhecida nos últimos anos, seu comportamento frente a uma infecção in vivo ainda não foi completamente compreendido. Neste trabalho nós analisamos a expressão dos TLR-2 e TLR-4 em células apresentadoras de antígeno em recém-nascidos com e sem sepse. CAUSUÍSTICA E MÉTODO: Trata-se de um estudo prospectivo realizado no período entre fevereiro de 2011 e janeiro de 2013 onde foram incluídos quarenta e cinco recém-nascidos a termo, sem malformação congênita, admitidos na Unidade de Cuidados Intensivos Neonatal do Instituto da Criança-HCFMUSP e divididos em grupos 1 e 2. O grupo 1 consistiu em 27 recém-nascidos com diagnóstico clínico e laboratorial de sepse tardia enquanto que o grupo 2 foi composto por 18 recém-nascidos sem quadro séptico vigente. As citocinas foram determinadas por teste de CBA em sangue periférico. A expressão e MFI dos TLR-2 e TLR-4 foi determinado por imunofenotipagem em APCs e linfócitos no sangue periférico total através de análise pelo citômetro de fluxo BD FACSDiva. RESULTADOS: Os dados clínicos foram semelhantes entre os grupos 1 e 2, exceto para o estado infeccioso. Microrganismos foram identificados em 37 % no grupo 1 e estes tiveram níveis mais elevados de citocinas pró-inflamatórias (IL-8, IL-6, IL-1beta) e de citocina anti-inflamatória (IL-10). Nas células dendríticas, a expressão de TLR-2 e 4 foi semelhante entre os grupos enquanto que houve menor expressão nos pacientes infectados da molécula co-estimuladora CD86 (p < 0,05) e expressão semelhante de CD1a e CD80 em relação aos RN não infectados. No monócito, o MFI para TLR-2 e a freqüência de expressão do TLR-4 foi maior no grupo 1 (p = 0,01). Apesar da frequência de linfócitos totais ter sido mais baixa no grupo 1 (p = 0,002), não foi observada diferença quanto as suas subpopulações exceto em relação a maior frequência de LT efetor no grupo infectado com menor expressão da molécula CD28. Houve maior frequência de LB ativados no grupo 1 enquanto que a população total e as demais subpopulações foram semelhantes em número, moléculas de ativação e na expressão dos TLR-2 e 4 em ambos os grupos. CONCLUSÃO: Este estudo analisou a resposta imune inata no recém-nascido com e sem sepse. As IL-6, IL-8 e IL-10 foram bons indicadores desta doença. Recém-nascidos sépticos, que dependem quase exclusivamente do sistema imune inato, apresentaram pouca resposta in vivo na ativação de células dendríticas e monócitos propiciando uma resposta imune deficiente e maior susceptibilidade à infecção / INTRODUCTION: Despite continuous progress in the clinical treatment and other supportive care therapies, sepsis remains a leading cause of morbidity and mortality in the intensive care unit with similar outcome throughout the past 50 years. The susceptibility to severe infection is partially due to newborn immature innate immune system associated to minimal in utero antigen exposure and effector T and B cell impaired function. Although the importance of pattern recognition domains such as Toll-like receptors (TLR) in the innate immune system activation has been fully acknowledged within the last few years its behavior in front of an in vivo infection scenario is still not completely understood. Here we analyzed the TLR-2 and TLR-4 expression in antigen-presenting cell in healthy and septic newborns. PATIENTS AND METHODS: This prospective study was conducted during the period from February 2011 until January 2013 at Sao Paulo University, Sao Paulo, Brazil. Forty-five term newborns without congenital malformation were included from the Newborn Intensive Care Unit at Children\'s Hospital. As group 1, 27 newborns who had clinical and laboratory diagnostic of late onset sepsis were included while 18 newborns were evaluated in a non-septic status and were included at group 2. Cytokines were measured by cytometric bead array in peripheral blood. TLR-2 and TLR-4 expression and MFI were determined by immunophenotyping at peripheral whole blood in APC cells and lymphocytes and analyzed on a BD FACSDiva flow cytometer. RESULTS: Clinical data was similar between septic and non-septic groups except for the infectious status. Group 1 had microorganisms identified in 37 % septic newborns associated with higher levels of pro-inflammatory (IL-8, IL-6, IL-1beta) and anti-inflammatory interleukins (IL-10). When it comes to dendritic cells, the expression of TLR-2 and 4 was similar between groups whereas there was lower expression of co-molecule CD86 (p < 0,05) and similar expression of CD1a and CD80 between infected and non-infected patients. At monocytes, the MFI for TLR-2 and the frequency of TLR-4 expression was higher in infected newborn (p=0,01). There were lower levels of total lymphocytes in infected patients (p=0,002) but no difference was observed in T cells subtypes frequency except for higher levels of effector T cell in infected group with lower expression of CD28 molecule. Group 1 had higher levels of activated B cell whereas total population and the other subsets were similar in number, activation molecules and TLR-2 and 4 expressions in both groups. CONCLUSION: This study investigated the innate immune response in septic and non-septic newborn. Interleukin levels 6, 8 and 10 were good indicators of sepsis. Septic newborns, which count most exclusively with innate immune system, had little in vivo response at dendritic cell and monocyte activation leading to an impaired immune response and increased susceptibility to infection

INTRODUÇÃO: Apesar do contínuo progresso no tratamento e suporte clínico a sepse continua sendo uma das principais causas de morbidade e mortalidade nas unidades de terapia intensiva, com desfechos semelhantes ao longo dos últimos 50 anos. A suscetibilidade à infecção grave no recém-nascido é parcialmente devida à imaturidade do sistema imune inato associado à mínima em exposição antigênica in utero e à ação ineficaz das células T efetoras e das célula B. Embora a ativação do sistema imune inato por padrões de reconhecimento (PRR) como os dos receptores Toll-like (TLR) tenham sua importância amplamente reconhecida nos últimos anos, seu comportamento frente a uma infecção in vivo ainda não foi completamente compreendido. Neste trabalho nós analisamos a expressão dos TLR-2 e TLR-4 em células apresentadoras de antígeno em recém-nascidos com e sem sepse. CAUSUÍSTICA E MÉTODO: Trata-se de um estudo prospectivo realizado no período entre fevereiro de 2011 e janeiro de 2013 onde foram incluídos quarenta e cinco recém-nascidos a termo, sem malformação congênita, admitidos na Unidade de Cuidados Intensivos Neonatal do Instituto da Criança-HCFMUSP e divididos em grupos 1 e 2. O grupo 1 consistiu em 27 recém-nascidos com diagnóstico clínico e laboratorial de sepse tardia enquanto que o grupo 2 foi composto por 18 recém-nascidos sem quadro séptico vigente. As citocinas foram determinadas por teste de CBA em sangue periférico. A expressão e MFI dos TLR-2 e TLR-4 foi determinado por imunofenotipagem em APCs e linfócitos no sangue periférico total através de análise pelo citômetro de fluxo BD FACSDiva. RESULTADOS: Os dados clínicos foram semelhantes entre os grupos 1 e 2, exceto para o estado infeccioso. Microrganismos foram identificados em 37 % no grupo 1 e estes tiveram níveis mais elevados de citocinas pró-inflamatórias (IL-8, IL-6, IL-1beta) e de citocina anti-inflamatória (IL-10). Nas células dendríticas, a expressão de TLR-2 e 4 foi semelhante entre os grupos enquanto que houve menor expressão nos pacientes infectados da molécula co-estimuladora CD86 (p < 0,05) e expressão semelhante de CD1a e CD80 em relação aos RN não infectados. No monócito, o MFI para TLR-2 e a freqüência de expressão do TLR-4 foi maior no grupo 1 (p = 0,01). Apesar da frequência de linfócitos totais ter sido mais baixa no grupo 1 (p = 0,002), não foi observada diferença quanto as suas subpopulações exceto em relação a maior frequência de LT efetor no grupo infectado com menor expressão da molécula CD28. Houve maior frequência de LB ativados no grupo 1 enquanto que a população total e as demais subpopulações foram semelhantes em número, moléculas de ativação e na expressão dos TLR-2 e 4 em ambos os grupos. CONCLUSÃO: Este estudo analisou a resposta imune inata no recém-nascido com e sem sepse. As IL-6, IL-8 e IL-10 foram bons indicadores desta doença. Recém-nascidos sépticos, que dependem quase exclusivamente do sistema imune inato, apresentaram pouca resposta in vivo na ativação de células dendríticas e monócitos propiciando uma resposta imune deficiente e maior susceptibilidade à infecção / INTRODUCTION: Despite continuous progress in the clinical treatment and other supportive care therapies, sepsis remains a leading cause of morbidity and mortality in the intensive care unit with similar outcome throughout the past 50 years. The susceptibility to severe infection is partially due to newborn immature innate immune system associated to minimal in utero antigen exposure and effector T and B cell impaired function. Although the importance of pattern recognition domains such as Toll-like receptors (TLR) in the innate immune system activation has been fully acknowledged within the last few years its behavior in front of an in vivo infection scenario is still not completely understood. Here we analyzed the TLR-2 and TLR-4 expression in antigen-presenting cell in healthy and septic newborns. PATIENTS AND METHODS: This prospective study was conducted during the period from February 2011 until January 2013 at Sao Paulo University, Sao Paulo, Brazil. Forty-five term newborns without congenital malformation were included from the Newborn Intensive Care Unit at Children\'s Hospital. As group 1, 27 newborns who had clinical and laboratory diagnostic of late onset sepsis were included while 18 newborns were evaluated in a non-septic status and were included at group 2. Cytokines were measured by cytometric bead array in peripheral blood. TLR-2 and TLR-4 expression and MFI were determined by immunophenotyping at peripheral whole blood in APC cells and lymphocytes and analyzed on a BD FACSDiva flow cytometer. RESULTS: Clinical data was similar between septic and non-septic groups except for the infectious status. Group 1 had microorganisms identified in 37 % septic newborns associated with higher levels of pro-inflammatory (IL-8, IL-6, IL-1beta) and anti-inflammatory interleukins (IL-10). When it comes to dendritic cells, the expression of TLR-2 and 4 was similar between groups whereas there was lower expression of co-molecule CD86 (p < 0,05) and similar expression of CD1a and CD80 between infected and non-infected patients. At monocytes, the MFI for TLR-2 and the frequency of TLR-4 expression was higher in infected newborn (p=0,01). There were lower levels of total lymphocytes in infected patients (p=0,002) but no difference was observed in T cells subtypes frequency except for higher levels of effector T cell in infected group with lower expression of CD28 molecule. Group 1 had higher levels of activated B cell whereas total population and the other subsets were similar in number, activation molecules and TLR-2 and 4 expressions in both groups. CONCLUSION: This study investigated the innate immune response in septic and non-septic newborn. Interleukin levels 6, 8 and 10 were good indicators of sepsis. Septic newborns, which count most exclusively with innate immune system, had little in vivo response at dendritic cell and monocyte activation leading to an impaired immune response and increased susceptibility to infection

In Enterobacteriaceae, the transcriptional regulator AmpR, a member of the LysR family, regulates the expression of a chromosomal β-lactamase AmpC. The regulatory repertoire of AmpR is broader in Pseudomonas aeruginosa, an opportunistic pathogen responsible for numerous acute and chronic infections including cystic fibrosis. Previous studies showed that in addition to regulating ampC, P. aeruginosa AmpR regulates the sigma factor AlgT/U and production of some quorum sensing (QS)-regulated virulence factors. In order to better understand the ampR regulon, the transcriptional profiles generated using DNA microarrays and RNA-Seq of the prototypic P. aeruginosa PAO1 strain with its isogenic ampR deletion mutant, PAO∆ampR were analyzed. Transcriptome analysis demonstrates that the AmpR regulon is much more extensive than previously thought influencing the differential expression of over 500 genes. In addition to regulating resistance to β-lactam antibiotics via AmpC, AmpR also regulates non-β-lactam antibiotic resistance by modulating the MexEF-OprN efflux pump. Virulence mechanisms including biofilm formation, QS-regulated acute virulence, and diverse physiological processes such as oxidative stress response, heat-shock response and iron uptake are AmpR-regulated. Real-time PCR and phenotypic assays confirmed the transcriptome data. Further, Caenorhabditis elegans model demonstrates that a functional AmpR is required for full pathogenicity of P. aeruginosa. AmpR, a member of the core genome, also regulates genes in the regions of genome plasticity that are acquired by horizontal gene transfer. The extensive AmpR regulon included other transcriptional regulators and sigma factors, accounting for the extensive AmpR regulon. Gene expression studies demonstrate AmpR-dependent expression of the QS master regulator LasR that controls expression of many virulence factors. Using a chromosomally tagged AmpR, ChIP-Seq studies show direct AmpR binding to the lasR promoter. The data demonstrates that AmpR functions as a global regulator in P. aeruginosa and is a positive regulator of acute virulence while negatively regulating chronic infection phenotypes. In summary, my dissertation sheds light on the complex regulatory circuit in P. aeruginosa to provide a better understanding of the bacterial response to antibiotics and how the organism coordinately regulates a myriad of virulence factors.

L’asplénie ou l’absence de la rate peut être congénitale, c’est- à -dire absente dès la naissance, ou bien acquise, par exemple lors d’une opération après un accident. L’asplénie congénitale est le plus souvent associée à d’autres problèmes développementaux. En particulier l’asplénie congénitale est associée à des problèmes de développement du cœur, dans le cadre des syndromes d’hétérotaxie. Ces syndromes d’hétérotaxie sont caractérisés par des problèmes de latéralité droite-gauche. Ainsi une personne ayant deux parties ‘droites’ n’aura pas de rate. A contrario, l’asplénie congénitale isolée est caractérisée par l’absence de rate et aucune autre malformation. L’asplénie congénitale isolée est une maladie très rare. Nous avons estimé la fréquence de la maladie à un cas pour un million de naissances. C’est aussi une maladie extrêmement mortelle. La grande majorité des patients ayant une asplénie congénitale isolée souffrent d’infections bactériennes sévères lors de l’enfance et la moitie des cas reportés sont décédés dus à une infection bactérienne, le plus souvent du à une infection par Streptococcus pneumoniae. Malgré la sévérité de cette maladie, celle-ci reste très peu connue et très peu étudiée. Ainsi le diagnostique est souvent trop tardif. Parmi les quelques dizaines de cas décrits dans la littérature, la moitié sont des cas familiaux avec plusieurs membres de la même famille affectée. Le mode de transmission semble être autosomique dominant dans la majorité des cas. En outre aucune preuve n’existe concernant un facteur environnemental pour cette maladie. Enfin des travaux récents ont montrés que l’absence de pancréas chez l’homme était une maladie génétique, et due à des mutations dans le gène GATA6 chez la moitié des patients. L’objectif de cette thèse est donc de déterminer l’origine génétique de l’asplénie congénitale isolée chez l’homme. J’ai fait l’hypothèse que l’asplénie congénitale isolée chez l’homme est due à des mutations mendéliennes dans un gène important pour le développement de la rate. Afin de tester notre hypothèse nous avons recruté des patients à travers des collaborations avec des médecins étrangers ainsi qu’un partenariat avec toutes les unités pédiatriques de France. Nous avons finalement pu recruter 37 patients appartenant à 24 familles différentes. La littérature sur le développement de la rate chez la souris et encore plus sur l’homme étant minimale, il était difficile d’identifier de bons gènes candidats pour être responsables de l’asplénie. Nous avons donc opté pour une stratégie portant sur le génome entier, sans biais lier a la littérature. La stratégie était d’utiliser le séquençage de l’exome de tous les patients. Le séquençage de l’exome est en fait le séquençage de tous les exons du génome, ou au moins 90% des exons du génome. La technique du séquençage de l’exome est arrivée à la fin de l’année 2009 et nous avons été un des premiers laboratoires à l’utiliser. Il fallait donc que nous l’essayons en premier sur un cas facile afin de vérifier que cette technique fonctionnait. Nous avons donc fait une étude préliminaire sur un cas ‘facile’. Par cas facile, il faut comprendre un cas où la probabilité que ce soit une mutation mendélienne dans un gène qui soit responsable de la maladie soit la plus forte possible, et où le nombre de gènes à regarder soit le plus faible possible. Un cas ‘facile’ est donc le cas d’une famille avec de nombreux patients, et de surcroit une famille consanguine. Dans le cas d’une famille consanguine la probabilité que ce soit une mutation récessive qui soit responsable de la maladie génétique est très importante. On peut alors se restreindre à analyser les régions du génome ou toutes les variations sont homozygotes. Nous avions une famille dans ce cas. Il y avait 4 patients dans cette famille souffrant d’infections bactériennes sévères dues à une asplenie fonctionnelle, ainsi que d’infections virales / Isolated congenital asplenia (ICA) is a rare primary immunodeficiency, first described in 1956, thattypically manifests in childhood with sudden, life-threatening, invasive bacterial disease. Patients withICA do not display any other overt developmental anomalies. The genetic etiology of ICA has remainedelusive. I hypothesized that ICA results from single-gene inborn errors of spleen development. I aimedto decipher the molecular genetic basis of ICA by pursuing a genome-wide approach, based on thesequencing of the whole-exome and the detection of copy number variations in all patients of ourcohort. I found that heterozygous mutations in RPSA, ribosomal protein SA, were present in more thanhalf of ICA patients (19/33). I then showed that haploinsufficiency of RPSA led to ICA in one kindredat least. RPSA is a protein involved in pre-rRNA processing and is an integral part of the ribosome. Thechallenge is, now, to understand the pathogenesis of the disease. How does a mutation in a ubiquitousand highly expressed gene lead to a spleen specific phenotype? This discovery will set the basis for abroader understanding of the development of the spleen in humans and the function of a ribosomalprotein. This discovery will also be beneficial to the families of patients with ICA, guiding geneticcounseling. It will lead to prevention of infections in newborns with mutations in RPSA. Finally themethod we used to analyze the exomes of the ICA cohort will be useful to discover the genetic etiologyof other genetic diseases.

Enterohemorrhagic Escherichia coli (EHEC) colonizes the intestine and produces the phage-encoded Shiga toxin (Stx) which is absorbed systemically and can lead to hemolytic uremic syndrome (HUS) characterized by hemolytic anemia, thrombocytopenia, and renal failure. EHEC, and two related pathogens, Enteropathogenic E. coli (EPEC), and the murine pathogen, Citrobacter rodentium, are attaching and effacing (AE) pathogens that intimately adhere to enterocytes and form actin “pedestals” beneath bound bacteria. The actin pedestal, because it is a unique characteristic of AE pathogens, has been the subject of intense study for over 20 years. Investigations into the mechanism of pedestal formation have revealed that to generate AE lesions, EHEC injects the type III effector, Tir, into mammalian cells, which functions as a receptor for the bacterial adhesin intimin. Tir-intimin binding then triggers a signaling cascade leading to pedestal formation. In spite of these mechanistic insights, the role of intimin and pedestal formation in EHEC disease remains unclear, in part because of the paucity of murine models for EHEC infection. We found that the pathogenic significance of EHEC Stx, Tir, and intimin, as well as the actin assembly triggered by the interaction of the latter two factors, could be productively assessed during murine infection by recombinant C. rodentium expressing EHEC virulence factors. Here we show that EHEC intimin was able to promote colonization of C. rodentium in conventional mice. Additionally, previous in vitro data indicates that intimin may have also function in a Tir-independent manner, and we revealed this function using streptomycin pre-treated mice. Lastly, using a toxigenic C. rodentium strain, we assessed the function of pedestal formation mediated by Tir-intimin interaction and found that Tir-mediated actin polymerization promoted mucosal colonization and a systemic Stx-mediated disease that shares several key features with human HUS.

Sepsis is the leading cause of death among critically ill patients and the 10th most common cause of death overall in the United States. The mortality rates increase with severity of the disease, ranging from 15% for sepsis to 60% for septic shock. Patient with sepsis can present varied clinical symptoms depending on the personal predisposition, causal microorganism, organ system involved, and disease severity. To facilitate sepsis diagnosis, the first sepsis consensus definitions was published in 1991 and then updated in 2001. Early recognition of a sepsis patient followed with timely and appropriate treatment and management strategies have been shown to significantly reduce sepsis-related mortality, and allows care to be provided at lower costs. Despite the rapid progress in the knowledge of pathophysiological mechanisms of sepsis and its treatment in the last two decades, identifying patient with sepsis and therapeutic approaches to sepsis and its complications remains challenging to critical care clinicians. Hence, the objectives of this thesis were to 1) evaluate the test characteristics of the two sepsis consensus definitions and delineate the differences in patient profile among patients meeting or not meeting sepsis definitions; 2) determine the relationship between the changes in several physiological parameters before sepsis onset and sepsis, and to determine whether these parameters could be used to identify sepsis in critically ill adults; 3) evaluate the effect of corticosteroids therapy on patient mortality. Data used in this thesis were prospectively collected from an electronic medical record system for all the adult patients admitted into the seven critical care units (ICUs) in a tertiary medical center. Besides analyzing data at the ICU stay level, we investigated patient information in various time frames, including 24-hour, 12-hour, and 6-hour time windows. In the first study of this thesis, the 1991 sepsis definition was found to have a high sensitivity of 94.6%, but a low specificity of 61.0%. The 2001 sepsis definition had a slightly increased sensitivity but a decreased specificity, which was 96.9% and 58.3%, respectively. The areas under the ROC curve for the two consensus definitions were similar, but less than optimal. The sensitivity and area under the ROC curve of both definitions were lower at the 24-hour time window level than those of the unit stay level, though the specificity increased slightly. At the time window level, the 1991 definitions performed slightly better than the 2001 definition. In the second study, minimum systolic blood pressure performed the best, followed by maximum respiratory rate in discriminating sepsis patients from SIRS patients. Maximum heart rate and maximum respiratory rate can differentiate sepsis patients from non-SIRS patients fairly well. The area under ROC of the combination of five physiological parameters was 0.74 and 0.90 for comparing sepsis to non-infectious SIRS patients and comparing sepsis to non-SIRS patients, respectively. Parameters typically performed better in 24-hour windows compared to 6-hour or 12-hour windows. In the third study, significantly increased hospital mortality and ICU mortality were observed in the group treated with low-dose corticosteroids than the control group based on the propensity score matched comparisons, and multivariate logistic regression analyses after adjustment for propensity score alone, covariates, or propensity score (in deciles) and covariates. This thesis advances the existing knowledge by systemically evaluating the test characteristics for the 1991 and 2001 sepsis consensus definitions, delineating physiological signs and symptoms of deterioration in the preceding 24 hours prior to sepsis onset, assessing the prediction performances of single or combined physiological parameters, and examining the use of corticosteroids treatment and survival among septic shock patients. In addition, this thesis sets an innovative example on how to use data from electronic medical records as these surveillance systems are becoming increasingly popular. The results of these studies suggest that a more parsimonious set of definitional criteria for sepsis diagnosis are needed to improve sepsis case identification. In addition, continuously monitored physiological parameters could help to identify patients who show signs of deterioration prior to developing sepsis. Last but not least, caution should be used when considering a recommendation on the use of low dose corticosteroids in clinical practice guidelines for the management of sepsis.

Yersinia pestis (Y. pestis), as the causative agent of plague, has caused deaths estimated to more than 200 million people in three historical plague pandemics, including the infamous Black Death in medieval Europe. Although infection with Yersinia pestis can mostly be limited by antibiotics and only 2000-5000 cases are observed worldwide each year, this bacterium is still a concern for bioterrorism and recognized as a category A select agent by the Centers for Disease Control and Prevention (CDC). The investigation into the host-pathogen interactions during Y. pestis infection is important to advance and broaden our knowledge about plague pathogenesis for the development of better vaccines and treatments. Y. pestis is an expert at evading innate immune surveillance through multiple strategies, several mediated by its type three secretion system (T3SS). It is known that the bacterium induces rapid and robust cell death in host macrophages and dendritic cells. Although the T3SS effector YopJ has been determined to be the factor inducing cytotoxicity, the specific host cellular pathways which are targeted by YopJ and responsible for cell death remain poorly defined. This thesis research has established the critical roles of caspase-8 and RIP kinases in Y. pestis-induced macrophage cell death. Y. pestis-induced cytotoxicity is completely inhibited in RIP1-/- or RIP3-/-caspase-8-/- macrophages or by specific chemical inhibitors. Strikingly, this work also indicates that macrophages deficient in either RIP1, or caspase-8 and RIP3, have significantly reduced infection-induced production of IL-1β, IL-18, TNFα and IL-6 cytokines; impaired activation of NF-κB signaling pathway and greatly compromised caspase-1 processing; all of which are critical for innate immune responses and contribute to fight against pathogen infection. Y. pestis infection causes severe and often rapid fatal disease before the development of adaptive immunity to the V bacterium, thus the innate immune responses are critical to control Y. pestis infection. Our group has previously established the important roles of key molecules of the innate immune system: TLR4, MyD88, NLRP12, NLRP3, IL-18 and IL-1β, in host responses against Y. pestis and attenuated strains. Yersinia has proven to be a good model for evaluating the innate immune responses during bacterial infection. Using this model, the role of caspase-8 and RIP3 in counteracting bacterial infection has been determined in this thesis work. Mice deficient in caspase-8 and RIP3 are very susceptible to Y. pestis infection and display reduced levels of pro-inflammatory cytokines in spleen and serum, and decreased myeloid cell death. Thus, both in vitro and in vivo results indicate that caspase-8 and RIP kinases are key regulators of macrophage cell death, NF-κB and caspase-1 activation in Yersinia infection. This thesis work defines novel roles for caspase-8 and RIP kinases as the central components in innate immune responses against Y. pestis infection, and provides further insights to the host-pathogen interaction during bacterial challenge.